PROJECT SUMMARY Disruption of the endothelial monolayer plays an integral role in the initiation of vascular inflammation, which leads to many cardiovascular diseases. Circulating monocytes adhere to vascular endothelial cells (EC) at injured area and migrate into subendothelial layer. Liver X Receptor (LXR) plays an important role in maintaining vascular homeostasis including the suppression of vascular inflammatory responses; however, its role in EC was unknown. In our recent study, we found that LXR?, a dominant isoform of LXR in vasculature, promotes EC migration and reendothelialization after vascular injury through activating the non-transcriptional action of estrogen receptor (ER) ?. We also have evidence that in EC a subpopulation of LXR? localizes in plasma membrane (PM) caveolae/lipid rafts, and that LXR? directly binds to and induces ER? phosphorylation via Akt signaling, thereby LXR? activates the non-nuclear signaling by ER?. Our OVERALL OBJECTIVE is to identify the mechanisms of non-nuclear LXR? function in vascular EC through the interaction with ER?, and also to examine how non-nuclear LXR?-ER? interaction plays important roles in the prevention of vascular diseases. Aim 1 is to identify the mechanism by which non-nuclear LXR? activates ER?. Whether the direct binding of LXR? to ER? in PM is necessary for the non-nuclear LXR activation via ER? will be tested. In addition, the molecular basis that LXR agonists stabilize the LXR?-ER? interaction will be tested. Aim 2 is to determine the role of non-nuclear LXR? function in EC function and vascular inflammatory responses. We will segregate non-nuclear from nuclear effects by the LXR?-ER? interaction in EC using newly generated mutant ER? knock-in mice that lack non-nuclear but retain nuclear function of ER?. We will also examine the effect by LXR? activation on EC expression of inflammatory cytokines and cell adhesion molecules as a potential non- nuclear LXR function. The effect of endothelial non-nuclear LXR activation on the monocyte adhesion to EC will be also tested in cell culture and in vivo. Aim 3 is to evaluate the utility of rhodium-LXR agonist conjugates to investigate non-nuclear action of LXR?. Newly synthesized compound that contain four molecules of GW3965 attached to a rhodium metal center will be examined as a novel tool for non-nuclear-specific LXR activation. Upon completion of this project, we hope to show that non-nuclear crosstalk between LXR? and ER? is important in vascular EC function, and to further our understanding of the mechanisms by which LXR? utilizes non-nuclear ER? signaling. Furthermore, this proposed research project has a potential to establish a novel therapeutic approach by improving vascular non-nuclear LXR? function without detrimental effects observed in the systemic LXR activation.